This proposal focuses on the dynamin-family large (~100kDa) GTPases that play critical roles in membrane scission events during cellular membrane trafficking, mitochondrial fission, and elsewhere. The functions of dynamin isoforms are central in intracellular trafficking, signaling, neuronal function, etc, impacting processes affected in many human diseases. Most recently, mutations in the pleckstrin homology (PH) domain of dynamin (one focus of our studies) were found to cause Charcot-Marie-Tooth disease, a hereditary progressive neuropathy. The dynamin relatives involved in mitochondrial fission (DRP1 in human, Dnmlp in yeast) play a key role in apoptosis, control of which is an important clinical goal. Thus, understanding the mechanisms of these GTPases is important for developing approaches to control apoptosis, cancer, neuronal degeneration, and many other diseases. Dynamin family members share 3 common domains: an N-terminal GTPase domain, a 'middle' domain, and a GTPase-effector domain (GED). They undergo cycles of assembly and disassembly regulated by GTP binding and hydrolysis. It is believed that this cycling is linked to the scission of membranes at precise locations, although how it is linked remains a subject of intense debate. In addition to the common domains, each dynamin family member contains unique domains likely to target the protein to its specific location and/or to link its GTPase activity to membrane scission. Dynamin has a pleckstrin homology (PH) domain and a proline/arginine-rich domain (PRO). DRP1 and Dnmlp have a distinct 'Drp' or 'insert B' domain that replaces the PH domain. Our focus is to understand the mechanistic role of these domains. Although the PH domain of dynamin binds phosphoinositides, and this is important for its function, our preliminary data suggest that it does not act as a membrane targeting module like other PH domains. Instead, dynamin appears to cluster Ptdlns(4,5)P2 in membranes (through its PH domain), providing a possible mechanism for dynamin's involvement in actin nucleation. Modulating lipid distribution in this way may reflect an 'effector' function for the PH domain of dynamin (and perhaps equivalent domains in other family members), which we propose to investigate. Combining cellular and in vitro biochemical/biophysical approaches, we propose the following specific aims: 1. To test the hypothesis that the dynamin PH domain functions as an effector, rather than a targeting, domain - transiently modulating Ptdlns(4,5)P2 density. We will also investigate the mechanisms through which dynamin promotes actin nucleation in a Ptdlns(4,5)P2-dependent manner. 2. To test the hypothesis that cardiolipin plays a critical role in Dnm1p/DRP1 function by binding to the Drp domain. We will also investigate the structural basis for cardiolipin recognition. Together, these studies promise to provide valuable insight into the mechanisms of action of these important large GTPases. [unreadable] [unreadable] [unreadable]